Rapid uptake of the neurotransmitter norepinephrine (NE) by the norepinephrine transporter (NET) regulates the magnitude and duration of neuronal signaling at monoamine synapses in the brain and in the peripheral nervous system. A large body of literature certifies that pharmacological modulation of neurotransmitter transporters has profound clinical effects. The necessity for precise control of NETs is evident, and their malfunction can be lethal. During ischaemia, NETs export NE out of sympathetic terminals in a nonvesicular mode of release and thereby contribute to potentially fatal arrhythmia; cardiovascular complications following NET blockade by tricyclic antidepressants and cocaine are well recognized. Yet we have little knowledge of how re-uptake occurs at the molecular level, what regulates it, or how transporter antagonists such as antidepressants and cocaine act to inhibit uptake. The studies we propose are virtually impossible to perform in the nerve terminals themselves. By studying NE uptake using cloned transporters in transfected mammalian cells, we can begin to understand how NETs control the magnitude and duration of neuronal signaling at synapses. Human NETs have now been cloned and the molecular details of transporter structure and function are emerging along several fronts. cDNA clones have been transfected into non-neuronal hosts where they have been used for radioligand uptake assays, binding studies, and immunological characterization. However, except for our preliminary work, no voltage- clamp analysis of NETs expressed either in oocytes or in mammalian cells has been done, and, to date, the study of NE transport mediated by NETs relies exclusively on radiolabled uptake assays. Specific hNET antibodies have helped us establish stable cell lines and to quantify expression and uptake in these model systems. The availability of mammalian cell lines for NETs with high levels of expression has allowed us to begin a methodical biophysical study of the functional states of transporters and to correlate rapid kinetic properties with transporter structure. The major aims of the proposal are (1) to study whole-cell currents under voltage clamp in hNET stably-and transiently- transfected cells over a wide range of mechanisms of voltage regulation using selected mutants with important residues already delineated, (3) to measure transporter currents in isolated patches and to correlate these with whole-cell currents, and (4) to investigate the cytoplasmic regulation of transporters by intracellular ions, particularly Ca. These studies will provide basic information about presynaptic regulation of NE transporters, loss of function that may result from improper regulation, and novel strategies for pharmacological intervention relevant to disease.